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Biochemical Pharmacology (v.71, #9)
A role for AMPA receptors in mood disorders by Andrew Alt; Eric S. Nisenbaum; David Bleakman; Jeffrey M. Witkin (pp. 1273-1288).
Major antidepressant agents increase synaptic levels of monoamines. Although the monoamine hypothesis of depression remains a cornerstone of our understanding of the pathophysiology of depression, emerging data has suggested that the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subtype of glutamate receptor may also play a pivotal role in depression. Positive allosteric modulators of AMPA receptors increase brain levels of brain-derived neurotrophic factor (BDNF) that impacts the viability and generation of neurons in key brain structures. AMPA receptor potentiators are active in rodent models predictive of antidepressant efficacy. The mechanisms by which AMPA receptor potentiators produce these biological effects, however, are uncertain. Current evidence points to an antidepressant mechanism that is independent of monoaminergic facilitation that is driven by neurogenesis, a process facilitated by increased BDNF expression. However, alternative hypotheses need to be considered given uncertainties in the relationship between BDNF increases and the effects of conventional antidepressant medications. Electrophysiological and protein conformational data indicate that structural variants of AMPA receptor potentiators can differentially modulate AMPA receptor-mediated currents, although the manner in which this impacts antidepressant efficacy is yet to be understood. Conventional antidepressants such as fluoxetine positively modulate AMPA receptors. This potentiation is engendered by specific phosphorylation pathways activated through the dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32). Other novel compounds with antidepressant-like effects in rodents may also produce their in vivo effects through potentiation of AMPA receptors. Thus, AMPA receptor potentiation might be a general mechanism through which the clinical outcome of antidepressant efficacy is achieved.
Keywords: Abbreviations; AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; cAMP; adenosine 3′,5′-cyclic monophosphate; CNQX; 6-cyano-7-nitroquinoxaline-2,3-dione; CX516; piperidine, 1-(6-quinoxalinylcarbonyl)-(9CI), Ampalex, BDP 12; CX691; piperidine, 1-(2,1,3-benzoxadiazol-5-ylcarbonyl)-(9CI), Farampator, Org 24448; DARPP-32; dopamine- and cAMP-regulated phosphoprotein of; M; r; 32,000; DNQX; 6,7-dinitroquinoxaline-2,3-dione; GCPR; G-protein-coupled receptor; GYKI 53655; 7; H; -1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-; N; ,8-dimethyl-monohydrochloride-(9CI); LY392098; N; -2-(4-(3-thienyl)phenyl)propyl 2-propanesulfonamide; LY404187; N; -2-(4-(4-cyanophenyl)phenyl)propyl 2-propanesulfonamide; LY451646; N; -[2-(4′-cyano[1,1′-biphenyl]-4-yl)propyl]-(9CI); LY503430; 4′-[(1R)-1-fluoro-1-methyl-2-[[(1-methylethyl)sulfonyl]amino]ethyl]-; N; -methyl-(9CI); MGS0039; 2-amino-3-[(3,4-dichlorophenyl)methoxy]-6-fluoro-, (1R,2R,3R,5R,6R)-(9CI); NMDA; N; -methyl-; d; -aspartateAMPA receptor potentiators; Brain-derived neurotrophic factor (BDNF); DARPP-32; Depression; GLU; A1; TrkB
Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro by Sam Maher; Siobhán McClean (pp. 1289-1298).
Antimicrobial peptides (AMPs) are a diverse group of proteinaceous compounds ranging in size, complexity and antimicrobial spectrum. The activity of AMPs against gut pathogens warrants the study of the interaction of AMPs with the mammalian gastrointestinal tract. In particular, the investigation of the in vitro cytotoxicity of these peptides is critical before they can be considered in clinical infections. The cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin was investigated in two gastrointestinal cell models (HT29 and Caco-2) with the MTT conversion assay, neutral red dye uptake assay and compared with that of vancomycin. The hemolytic activities were also investigated in sheep erythrocytes and the effect of AMPs on paracellular permeability was examined by transepithelial resistance (TEER) and TEM. Gallidermin was the least cytotoxic AMP followed by nisin A, magainin I, magainin II and melittin. Melittin and nisin were the only peptides to result in significant hemolysis. However, while nisin caused hemolysis at concentrations which were 1000-fold higher than those required for antimicrobial activity, melittin was hemolytic at concentrations in the same order of magnitude as its antimicrobial activity. Melittin was the only AMP to affect paracellular permeability. Long term melittin treatment also resulted in loss of microvilli, an increase in cell debris and destruction of intestinal tight junctions and cell–cell adhesion. Gallidermin shows most promise as a therapeutic agent, with relatively low cytotoxicity and potent antimicrobial activities. Melittin, while showing little potential as an antimicrobial agent, may have potential in delivery of poorly bioavailable drugs.
Keywords: Antimicrobial peptide; Cytotoxicity; Intestinal epithelial cells; Gastrointestinal tract infections; Epithelial integrity; Transepithelial resistance
The voltage dependence of recovery from use-dependent block by QX-222 separates mechanisms for drug egress in the cardiac sodium channel by Harvey A. Lardin; Peter J. Lee (pp. 1299-1307).
In neuronal sodium channels of squid giant axons, recovery from QX-222 block is slowed by hyperpolarization. However, in ventricular cells, hyperpolarization speeds recovery. Previously, we showed that isoform-specific residues in the external side of the cardiac sodium channel isoform (D1P-loop C373 and D4S6 T1752) influence use-dependent block (UDB) by lidocaine. To determine whether these isoformspecific residues contribute to the contrasting voltage-dependent recovery observed in ventricular myocytes, we measured recovery rates from UDB by QX-222 at holding potentials of 120, 140, 160 and 180mV for wild-type cardiac channel (WT), the mutants C373Y (CY) and T1752V (TV), and C373Y/T1752V (CY/TV). Unlike neuronal channels, cardiac sodium channels recovered from QX block faster at hyperpolarized potentials. All mutations slowed QX-222 recovery, with the greatest rate reduction observed for the double mutant, indicating that the isoform-specific residues define external drug paths. The recovery rates varied linearly with voltage over the range tested, and we used the slopes of rate versus voltage plots to quantify voltage dependence. The TV mutation caused reduction in recovery rates without changing the slope, indicating that the mutation closed a voltage-independent egress path. The CY mutation, however, flattened the slope and reduced the voltage dependence of recovery. In addition, the reduction in rate caused by CY/TV is less than the sum of those for CY and TV, suggesting that the impacts of these two residues are interrelated. Therefore, we propose that the isoform-specific residues C373 and T1752 change recovery from UDB by distinct mechanisms but determine a common drug egress path.
Keywords: Voltage-gated sodium channel; Local anesthetic drug; QX-222; Use-dependent block; Voltage dependence of recovery; Isoform-specific residues
Synthetic peroxisome proliferator-activated receptor γ agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cells by Adrian T. Coyle; B. Therese Kinsella (pp. 1308-1323).
The human thromboxane (TX)A2 receptor (TP) gene encodes two TP isoforms, TPα and TPβ, that are regulated by distinct promoters designated promoter Prm1 and Prm3, respectively. Previous studies established that 15d-Δ12,14-prostaglandin J2 (15d-PGJ2) selectively inhibits Prm3 activity and TPβ expression through a peroxisome proliferator-activated receptor (PPAR)γ mechanism without affecting Prm1 activity or TPα expression in human megakaryocytic erythroleukemia (HEL) 92.1.7 cells. Herein, we investigated the effect of synthetic thiazolidinedione (TZD) PPARγ ligands rosiglitazone and troglitazone on TP gene expression in HEL cells. Like 15d-PGJ2, both TZDs suppressed Prm3 activity, TPβ mRNA expression and TP-mediated calcium mobilization without affecting Prm1 or TPα mRNA expression. However, unlike 15d-PGJ2, both TZDs mediated their PPARγ-dependent effects through trans-repression of an activator protein-1 (AP-1) element, a site previously found to be critical for basal Prm3 activity. These data provide further evidence for the role of PPARγ in regulating the human TP gene; they highlight further differences in TPα and TPβ expression/regulation and point to essential differences between natural and synthetic PPARγ agonists in mediating those effects.
Keywords: Abbreviations; AP-1; activator protein-1; 15d-PGJ; 2; 15-deoxy (Δ); 12,14; -prostaglandin J; 2; E; exon; EMSA; electromobility shift assay; FBS; foetal bovine serum; HEK; human embryonic kidney; HEL; human erythroleukemia; I; intron; NT; nucleotide; PG; prostaglandin; P; promoter; PMA; phorbol myristic acid; PPAR; peroxisome proliferator-activated receptor; PPRE; peroxisome proliferator-activated receptor response element; Prm; promoter; RLU; relative luciferase units; RXR; retinoic acid X receptor; TP; thromboxane receptor; TI; transcription initiation; TXA; 2; thromboxane A; 2; UTR; untranslated region; VSM; vascular smooth muscle cellThromboxane receptor; Isoforms; Promoter; Peroxisome proliferator-activated receptor Δ; Thiazolidinedione; 15-Deoxy-Δ; 12,14; -prostaglandin J; 2
Hydroxymethylglutaryl-coenzyme A reductase inhibitors induce apoptosis in human cardiac myocytes in vitro by Svitlana Demyanets; Christoph Kaun; Stefan Pfaffenberger; Philipp J. Hohensinner; Gersina Rega; Johannes Pammer; Gerald Maurer; Kurt Huber; Johann Wojta (pp. 1324-1330).
Recent findings have implicated hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors or statins, an established class of drugs for the treatment of hypercholesterolemia, in tissue remodeling in the heart. Statins induce apoptosis in different cell culture systems including rat neonatal cardiomyocytes. We investigated possible effects of different statins in vitro in human adult cardiac myocytes on the expression of proteins thought to be involved in the regulation of apoptosis such as Mcl-1, an inhibitor of apoptosis, Bax, an inducer of apoptosis, as well as on cytoplasmic histone-associated-DNA-fragments. Human adult cardiac myocytes (HACM) were treated with different statins at concentrations from 0.01 to 5μM for up to 96h. Whereas the lipophilic statin simvastatin at a concentration of 5μM downregulated Mcl-1 mRNA by 49%, the hydrophilic pravastatin had no effect. Bax mRNA levels were not affected by neither of the statins. Simvastatin but not pravastatin reduced Mcl-1 protein expression whereas Bax protein was not detectable in HACM as determined by Western blotting. Simvastatin, atorvastatin and fluvastatin induced an up to seven-fold increase in histone-associated-DNA-fragments whereas pravastatin did not. Simvastatin up regulated histone-associated-DNA-fragments dose-dependently, and mevalonate and geranylgeranyl pyrophosphate reversed this effect to control levels. Our results show that lipophilic statins can induce a pro-apoptotic state in human adult cardiac myocytes in vitro. We speculate that, similar to findings in animal models, statins might be involved in the attenuation of cardiac hypertrophy and remodeling in humans by modulating the balance between cell survival and apoptosis.
Keywords: Statins; Cardiac myocytes; Apoptosis; Cardiac remodeling; Hypertrophy
Involvement of tumor necrosis factor (TNF)-α in phorbol ester 12- O-tetradecanoylphorbol-13-acetate (TPA)-induced skin edema in mice by Masao Murakawa; Kumiko Yamaoka; Yoshitaka Tanaka; Yoshiaki Fukuda (pp. 1331-1336).
Topical application of 12- O-tetradecanoylphorbol-13-acetate (TPA) to mouse ear induced a prolonged skin inflammation. Histological analysis revealed that the early stage (∼3h) and later stage (6–24h) of the skin reaction are characterized by dermal edema and cell accumulation, respectively. Topical application with TPA also induced increase in the level of TNF-α and prostagrandin E2 (PGE2) at the application site. The increase of TNF-α was transient with a peak at ∼5h, followed by a gradual elevation of PGE2 level in the skin. An in vitro study with human keratinocytes as well as immunohistochemical analysis suggested that TNF-α induction in the skin might be produced by epidermis treated with TPA. Administration of a cyclooxygenase inhibitor indomethacin inhibited the later stage of the TPA-induced edema. In contrast, TNF-α antagonist etanercept inhibited exclusively the early stage of the reaction. Taken together, these data demonstrate that the prolongation of the skin inflammation induced by TPA may be due to the sequential production of proinflammatory mediators such as eicosanoids and cytokines, and show for the first time the importance of TNF-α in the TPA-induced dermatitis especially at the stage where dermal edema is significant.
Keywords: Mouse; TPA; Skin inflammation; TNF; Eicosanoid
Hydroxyl radical scavenging reactivity of proton pump inhibitors by Wolfgang Alexander Simon; Ernst Sturm; Hans-Jürgen Hartmann; Ulrich Weser (pp. 1337-1341).
In addition to the established control of acid secretion of the class of proton pump inhibitors (PPI) reactivity from the pyridyl methyl sulphinyl benzimidazole type a second independent anti-inflammatory reactivity was observed in vitro. This inhibitory reactivity was clearly noticed using three different assays where the aggressive hydroxyl radicals were successfully trapped in a concentration dependent manner. There is unequivocal evidence that the proton pump inhibitors having the sulphoxide group are able to scavenge hydroxyl radicals which are generated during a Fenton reaction. By way of contrast, the corresponding thioethers were substantially less active. No detectable effect was seen in the superoxide radical scavenging system. In conclusion, pantoprazole as well as the other proton pump inhibitors have a pronounced inhibitory reactivity towards hydroxyl radicals.
Keywords: Proton pump inhibitors; Pantoprazole; Hydroxyl radical; Hydroxyl radical scavenger
3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity by Matthew D. Lynx; Alice T. Bentley; Edward E. McKee (pp. 1342-1348).
3′-Azido-3′-deoxythymidine (AZT) is a staple of highly active antiretroviral therapy (HAART). Prior to HAART, long-term use of high-dosage AZT caused myopathy, cardiomyopathy, and hepatotoxicity, associated with mitochondrial DNA depletion. As a component of HARRT, AZT causes cytopenias and lipodystrophy. AZT-5′-triphosphate (AZTTP) is a known inhibitor of the mitochondrial polymerase γ and has been targeted as the source of the mitochondrial DNA depletion. However, in previous work from this laboratory with isolated rat heart mitochondria, AZT phosphorylation beyond AZT-5′-monophosphate (AZTMP) was not detected. Rather, AZT was shown to be a more potent inhibitor of thymidine phosphorylation (50% inhibitory concentration (IC50) of 7.0±1.0μM) than AZTTP is of polymerase γ (IC50 of >100μM), suggesting that depletion of mitochondrial stores of TTP may limit replication. This work has investigated whether an identical mechanism might account for the hepatotoxicity seen with long-term use of AZT. Isolated rat liver mitochondria were incubated with labeled thymidine or AZT, and the rate and extent of phosphorylation were determined by HPLC analysis of acid-soluble extracts of the incubated mitochondria. The results showed that in the phosphorylation of thymidine to TMP, liver mitochondria exhibit a higher Vmax and Km than heart mitochondria, but otherwise heart and liver mitochondria display similar kinetics. AZT is phosphorylated to AZTMP, but no further phosphorylated forms were detected. In addition, AZT inhibited the production of TTP, with an IC50 of 14.4±2.6μM AZT. This is higher, but comparable to, the results seen in isolated rat heart mitochondria.
Keywords: Abbreviations; AIDS; acquired immunodeficiency syndrome; AZT; 3′-azido-3′-deoxythymidine; AZTDP; 3′-azido-3′-deoxythymidine-5′-diphosphate; AZTMP; 3′-azido-3′-deoxythymidine-5′-monophosphate; AZTTP; 3′-azido-3′-deoxythymidine-5′-triphosphate; HAART; highly active antiretroviral therapy; HIV; human immunodeficiency virus; IC; 50; 50% inhibitory concentration; S.E.M.; standard error of meanAZT; Liver mitochondria; Thymidine; Mitochondrial toxicity; Thymidine kinase 2; Reverse transcriptase inhibitors
Organotin compounds enhance 17β-hydroxysteroid dehydrogenase type I activity in human choriocarcinoma JAr cells: Potential promotion of 17β-estradiol biosynthesis in human placenta by Tsuyoshi Nakanishi; Youhei Hiromori; Hideaki Yokoyama; Mihoko Koyanagi; Norio Itoh; Jun-Ichi Nishikawa; Keiichi Tanaka (pp. 1349-1357).
Organotin compounds, such as tributyltin (TBT) and triphenyltin (TPT), are typical environmental contaminants and suspected endocrine-disrupting chemicals because they cause masculinization in female mollusks. However, it remains unclear whether organotin compounds also cause crucial toxicities in human sexual development and reproductive functions. We investigated the effects of 17 tin compounds on the catalytic activity and mRNA expression of 17β-hydroxysteroid dehydrogenase type I (17β-HSD I) in human choriocarcinoma JAr cells. At nontoxic concentrations, both trialkyltins with propyl, butyl or cyclohexyl substituents on the tin atom and triphenyltin (TPT) enhanced 17β-HSD I mRNA transcription and enzyme activity in a dose-dependent fashion. Although tetraalkyltin compounds such as tetrabutyltin and tributylvinyltin also increased the mRNA expression and enzyme activity of 17β-HSD I, the concentrations necessary for activation were >30–100 times greater than those for trialkyltins. Inorganic tin had no effect on the catalytic activity and mRNA expression of 17β-HSD I. Interestingly, diphenyltin and monophenyltin, which are metabolites of TPT, enhanced 17β-HSD I activity with a concomitant increase in mRNA expression, whereas dibutyltin and monobutyltin, which are metabolites of tributyltin, enhanced 17β-HSD I activity without a concomitant increase in mRNA expression. These results suggest that organotin compounds are potent stimulators of 17β-estradiol biosynthesis to enhance 17β-HSD I activity in the human placenta in vitro; the placenta represents a potential target organ for these compounds, whose endocrine-disrupting effects might be the result of local changes in 17β-estradiol concentrations in pregnant women.
Keywords: Abbreviations; TBT; tributyltin; TPT; triphenyltin; 17β-HSD I; 17β-hydroxysteroid dehydrogenase type I; 17β-HSDs; 17β-hydroxysteroid dehydrogenases; E1; estrone; E2; 17β-estradiol; 9cRA; 9-; cis; retinoic acid; atRA; all-; trans; retinoic acid; FCS; fetal calf serum; MEM; minimal essential medium; RXR; retinoid X receptor; LG; LG100268; RAR; retinoic acid receptorOrganotin; Tributyltin; Triphenyltin; 17β-Hydroxysteroid dehydrogenase type I (17β-HSD I); Placenta; Estrogen; Retinoid X receptor (RXR)
Quaternary ammonium-linked glucuronidation of trans-4-hydroxytamoxifen, an active metabolite of tamoxifen, by human liver microsomes and UDP-glucuronosyltransferase 1A4 by Kenichiro Ogura; Yuko Ishikawa; Teppei Kaku; Takahito Nishiyama; Tomokazu Ohnuma; Kei Muro; Akira Hiratsuka (pp. 1358-1369).
Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. Trans-4-hydroxy-TAM ( trans-4-HO-TAM), one of the TAM metabolites in humans, has been considered to be an active metabolite of TAM because of its higher affinity toward estrogen receptors (ERs) than the parent drug and other side-chain metabolites. In the present study, we found a new potential metabolic pathway of trans-4-HO-TAM and its geometrical isomer, cis-4-HO-TAM, via N-linked glucuronic acid conjugation for excretion in humans. N+-Glucuronides of 4-HO-TAM isomers were isolated along with O-glucuronides from a reaction mixture consisting of trans- or cis-4-HO-TAM and human liver microsomes fortified with UDP-glucuronic acid and identified with their respective synthetic specimens by high performance liquid chromatography–electrospray ionization time-of-flight mass spectrometry. Although N- and O-glucuronidating activities of human liver microsomes toward trans-4-HO-TAM were nearly comparable, O-glucuronidation was predominant for cis-4-HO-TAM conjugation. Only UGT1A4 catalyzed the N-linked glucuronidation of 4-HO-TAM among recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. In contrast, all UGT isoforms, except for UGT1A3 and UGT1A4, catalyzed O-glucuronidation of 4-HO-TAM. Although O-glucuronidation of 4-HO-TAM greatly decreased binding affinity for human ERs, 4-HO-TAM N+-glucuronide still had binding affinity similar to 4-HO-TAM itself, suggesting that N+-glucuronide might contribute to the biological activity of TAM in vivo.
Keywords: Abbreviations; BSA; bovine serum albumin; ESI-TOF-MS; electrospray ionization time-of-flight mass spectrometry; ER; estrogen receptor; GA; glucuronic acid; HRP; horseradish peroxidase; 4-HO-TAM; 4-hydroxytamoxifen; HPLC; high performance liquid chromatography; metabolite Y; 1-[4-(2-hydroxyethoxy)phenyl]-1,2-diphenylbut-1(; Z; )-ene); TAM; tamoxifen; HFC; 7-hydroxy-4-(trifluoromethyl)coumarin; TLC; thin-layer chromatography; UDPGA; UDP-glucuronic acid; UGT; UDP-glucuronosyltransferaseTamoxifen; 4-Hydroxytamoxifen; UDP-glucuronosyltransferase; N; -Glucuronidation; Human liver microsomes; Estrogen receptor
N6-Methyl-AMP aminohydrolase activates N6-substituted purine acyclic nucleoside phosphonates by Markéta Schinkmanová; Ivan Votruba; Antonín Holý (pp. 1370-1376).
In this study we present the identification and characterization of the enzyme involved in the N6-cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine ( N6-cyclopropyl-PMEDAP) conversion to biologically active 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) as well as abacavir 5′-phosphate to carbovir 5′-phosphate. This enzyme was purified from rat liver to homogeneity; it appears to be composed from six 42kDa subunits and its native form has the molecular weight 260kDa. This so far unknown enzyme catalyzes conversion of both N6-methyl-AMP and N6-methyl-dAMP to IMP and/or dIMP, respectively. The enzyme acts as 6-( N-substituted amino)purine 5′-nucleotide aminohydrolase with the reaction mechanism very similar to AMP deaminase. The enzyme does not deaminate AMP and dAMP, or the corresponding nucleosides. It is inhibited by deoxycoformycin 5′-phosphate but not by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).
Keywords: Abbreviations; PMEDAP; 9-[2-(phosphonomethoxy)-ethyl]-2,6-diaminopurine; cypr-PMEDAP; N; 6; -cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine; me-PMEDAP; N; 6; -methyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine; me; 2; -PMEDAP; N; 6; -dimethyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine; cypr-PMEA; N; 6; -cyclopropyl-9-[2-(phosphonomethoxy)ethyl]adenine; me; 2-; PMEA; N; 6; -dimethyl-9-[2-(phosphonomethoxy)ethyl]adenine; PMEG; 9-[2-(phosphonomethoxy)ethyl]guanine; ABC-MP; abacavir 5′-monophosphate; CBV-MP; carbovir 5′-monophosphate; me-AMP; N; 6; -methyl-AMP; me; 2; -AMP; N; 6; -dimethyl-AMP; me-dAMP; N; 6; -methyl-dAMP; EHNA; erythro; -9-(2-hydroxy-3-nonyl)adenine; dCF; deoxycoformycin; dCF-MP; deoxycoformycin 5′-monophosphate N; 6; -Methyl-AMP aminohydrolase; N; 6; -Methyl-AMP; N; 6; -Cyclopropyl-2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine; 9-[2-(Phosphonomethoxy)ethyl]guanine; Abacavir 5′-monophosphate; Carbovir 5′-monophosphate
Oxidative metabolism of 5-methoxy- N, N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes by Shizuo Narimatsu; Rei Yonemoto; Keita Saito; Kazuo Takaya; Takuya Kumamoto; Tsutomu Ishikawa; Masato Asanuma; Masahiko Funada; Kimio Kiryu; Shinsaku Naito; Yuzo Yoshida; Shigeo Yamamoto; Nobumitsu Hanioka (pp. 1377-1385).
In vitro quantitative studies of the oxidative metabolism of (5-methoxy- N, N-diisopropyltryptamine, 5-MeO-DIPT, Foxy) were performed using human liver microsomal fractions and recombinant CYP enzymes and synthetic 5-MeO-DIPT metabolites. 5-MeO-DIPT was mainly oxidized to O-demethylated (5-OH-DIPT) and N-deisopropylated (5-MeO-IPT) metabolites in pooled human liver microsomes. In kinetic studies, 5-MeO-DIPT O-demethylation showed monophasic kinetics, whereas its N-deisopropylation showed triphasic kinetics. Among six recombinant CYP enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) expressed in yeast or insect cells, only CYP2D6 exhibited 5-MeO-DIPT O-demethylase activity, while CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 showed 5-MeO-DIPT N-deisopropylase activities. The apparent Km value of CYP2D6 was close to that for 5-MeO-DIPT O-demethylation, and the Km values of other CYP enzymes were similar to those of the low- Km (CYP2C19), intermediate- Km (CYP1A2, CYP2C8 and CYP3A4) and high- Km phases (CYP2C9), respectively, for N-deisopropylation in human liver microsomes. In inhibition studies, quinidine (1μM), an inhibitor of CYP2D6, almost completely inhibited human liver microsomal 5-MeO-DIPT O-demethylation at a substrate concentration of 10μM. Furafylline, a CYP1A2 inhibitor, quercetin, a CYP2C8 inhibitor, sulfaphenazole, a CYP2C9 inhibitor and ketoconazole, a CYP3A4 inihibitor (5μM each) suppressed about 60%, 45%, 15% and 40%, respectively, of 5-MeO-DIPT N-deisopropylation at 50μM substrate. In contrast, omeprazole (10μM), a CYP2C19 inhibitor, suppressed only 10% of N-deisopropylation by human liver microsomes, whereas at the same concentration the inhibitor suppressed the reaction by recombinant CYP2C19 almost completely. These results indicate that CYP2D6 is the major 5-MeO-DIPT O-demethylase, and CYP1A2, CYP2C8 and CYP3A4 are the major 5-MeO-DIPT N-deisopropylase enzymes in the human liver.
Keywords: Abbreviations; CYP; cytochrome P450; OR; NADPH-cytochrome P450 reductase; 5-MeO-DIPT; 5-methoxy-; N; ,; N; -diisopropyltryptamine; 5-MeO-IPT; 5-methoxy-; N; -ispropyltryptamine; 5-OH-DIPT; 5-hydroxy-; N; ,; N; -diisopropyltryptamine; G-6-P; glucose 6-phosphate; HPLC; high-performance liquid chromatography; LC/MS; liquid chromatography–mass spectrometry; PCR; polymerase chain reactionFoxy; 5-MeO-DIPT; 5-OH-DIPT; 5-MeO-IPT; CYP2D6; CYP1A2; CYP2C8; CYP3A4
Catalytic roles of CYP2D6.10 and CYP2D6.36 enzymes in mexiletine metabolism: In vitro functional analysis of recombinant proteins expressed in Saccharomyces cerevisiae by Nobumitsu Hanioka; Yoshihito Okumura; Yoshiro Saito; Hiroyuki Hichiya; Akiko Soyama; Keita Saito; Kazuyuki Ueno; Jun-ichi Sawada; Shizuo Narimatsu (pp. 1386-1395).
Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one-third of the medicines in current clinical use and exhibits genetic polymorphism with interindividual differences in metabolic activity. To precisely investigate the effect of CYP2D6*10B and CYP2D6*36 frequently found in Oriental populations on mexiletine metabolism in vitro, CYP2D6 proteins of wild-type (CYP2D6.1) and variants (CYP2D6.10 and CYP2D6.36) were heterologously expressed in yeast cells and their mexiletine p- and 2-methyl hydroxylation activities were determined. Both variant CYP2D6 enzymes showed a drastic reduction of CYP2D6 holo- and apoproteins compared with those of CYP2D6.1. Mexiletine p- and 2-methyl hydroxylation activities on the basis of the microsomal protein level at the single substrate concentration (100μM) of variant CYP2D6s were less than 6% for CYP2D6.10 and 1% for CYP2D6.36 of those of CYP2D6.1. Kinetic analysis for mexiletine hydroxylation revealed that the affinity toward mexiletine of CYP2D6.10 and CYP2D6.36 was reduced by amino acid substitutions. The Vmax and Vmax/ Km values of CYP2D6.10 on the basis of the microsomal protein level were reduced to less than 10% of those of CYP2D6.1, whereas the values on the basis of functional CYP2D6 level were comparable to those of CYP2D6.1. Although it was impossible to estimate the kinetic parameters for the mexiletine hydroxylation of CYP2D6.36, the metabolic ability toward mexiletine was considered to be poorer not only than that of CYP2D6.1 but also than that of CYP2D6.10. The same tendency was also observed in kinetic analysis for bufuralol 1″-hydroxylation as a representative CYP2D6 probe. These findings suggest that CYP2D6*36 has a more drastic impact on mexiletine metabolism than CYP2D6*10.
Keywords: Abbreviations; CYP; cytochrome P450; HPLC; high-performance liquid chromatography; SRS; substrate recognition siteMexiletine; CYP2D6; Genetic polymorphism; CYP2D6*10; CYP2D6*36